Hepatitis C virus (HCV) infection is a major health problem that leads to chronic liver disease, such as cirrhosis and hepatocellular carcinoma, in a substantial number of infected individuals, estimated to be 2-15% of the world's population. There are an estimated 4.5 million infected people in the United States alone, according to the U.S. Center for Disease Control. According to the World Health Organization, there are more than 200 million infected individuals worldwide, with at least 3 to 4 million people being infected each year. Once infected, about 20% of people clear the virus, but the rest can harbor HCV the rest of their lives. Ten to twenty percent of chronically infected individuals eventually develop liver-destroying cirrhosis or cancer. The viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles, or sexually and vertically from infected mothers or carrier mothers to their offspring. Current treatments for HCV infection, which are restricted to immunotherapy with recombinant interferon-α alone or in combination with the nucleoside analog ribavirin, are of limited clinical benefit. Moreover, there is no established vaccine for HCV. Consequently, there is an urgent need for improved therapeutic agents that effectively combat chronic HCV infection.
Purine phosphoramidates have been shown to be potent inhibitors of the HCV virus (U.S. patent application Ser. No. 12/053,015, see also WO 2008/121634). However, preparation of these compounds has been made difficult due to poor yields associated with the coupling of the ribose sugar to the purine base and because of poor C-1′ beta-stereoselectivity associated with the ribose to purine base coupling step.
Generally, there are two ways to prepare a nucleoside analogue. The first way follows a linear synthetic sequence in which the target nucleoside is prepared from an appropriate nucleoside. In this approach, usually there is less concern about stereoselective chemistry as most if not all of the stereocenters are set. However, the synthesis can be lengthy if extensive modification of the sugar is required.
An alternative approach toward the synthesis of novel nucleosides utilizes a convergent synthesis where a sugar portion is separately modified and later coupled with an appropriate silylated base (Vorbrueggen et al., J. Org. Chem. 1976, 41, 2084). In the case of ribose derivatives in which there is a 2-α-O-acyl group present, the desired β stereochemistry at the 1′-position is secured by neighboring group participation in the presence of a Lewis acid such as SnCl4 or TMSOTf. However, if the sugar has no 2-α-O-acyl group as for 2-deoxy nucleoside, the Vorbrueggen conditions would be expected to generate an isomeric mixture which is then often difficult to separate. A common way to avoid this stereochemical problem is to employ an α-halosugar so that an SN2 type coupling with a salt of a purine base or a silylated pyrimidine base would generate the desired β isomer enriched mixture (Kazimierczuk, Z. et al. J. Am. Chem. Soc. 1984, 106, 6379-6382; Chun, B. K. et al J. Org. Chem., 2000, 65, 685-693; Zhong, M. et al. J. Org. Chem. 2006, 71, 7773-7779). However, the main problem of this approach from a process chemistry point of view is that, in many cases, it is difficult to obtain the desired reactive α-halosugar in a good yield without any difficult purification steps. There are many literature and patent examples of reacting salts of purine bases with α-halosugars.
Another possible way to do an SN2 type coupling is enzymatic glycosylation in which the sugar-1-α-O-phosphate is coupled with purine base using either isolated enzymes or whole cells. The phosphate intermediate can be generated enzymatically from another nucleoside containing the desired sugar. This coupled reaction is called transglycosylation. This conversion is highly stereospecific. Unfortunately, natural enzymes only work with a limited number of modified sugars. For custom sugars, existing enzymes from a range of microorganisms need to be screened for activity or through extensive research there is a possibility that a mutated enzyme can be selected and produced though genetic engineering (Komatsu, H. et al Tetrahedron Lett. 2003, 44, 2899-2901; Okuyama, K. et al. Biosci. Biotechnol. Biochem. 2003, 67(5), 989-995). 2′-Fluorinated nucleosides are difficult to enzymatically glycosylate but it has been accomplished using specialized natural enzymes (Krenitsky et al., J. Med. Chem. 1993, 36, 119-12) or with proprietary genetically engineered enzymes (Metkinen Chemistry, Kuusisto, Finland). There is no literature report of using enzymatic glycosylation for the 2′-fluoro-2′-C-methyl sugar. If it were possible, it would be necessary to start with 2′-fluoro-2′-C-methyluridine for transglycosylation or the 1-O-α-phosphate of the sugar for glycosylation. The cost of the synthesis of these starting materials approaches the cost of the final purines made chemically by the proposed route.
A final alternative method to couple a sugar with a purine base is through the use of Mitsunobu chemistry. This approach uses a condensing reagent such as N,N-dicyclohexylcarbodiiomide (DCC) and triphenylphosphine. Although this reaction accepts a wide variety of substrates, yields are typically lower and there is no stereoselectivity. Purification of the product from the Mitsunubo reagents and by-products is often challenging as well.
2′-deoxy-2′-fluoro-2′-C-methyl purine nucleosides and their corresponding nucleotide phosphoramidates belong to the 2′-deoxy nucleoside category since there is no directing α-acyloxy group in 2′-position. A close derivative of the purine analogs was first prepared using the linear nucleoside route in a less than 5% overall yield due to the complexity of forming the 2′ quaternary center. The lowest yielding step, fluorination, was done late in the sequence. This route was unsuitable for large scale synthesis (Clark, J. L. et al. Bioorg. Med. Chem. Lett. 2006, 16, 1712-1715).